Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).

Nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid type A receptors (GABARs) are members of the pentameric ligand-gated ion channel superfamily. Drugs acting as positive allosteric modulators of muscle-type αβγδ nAChRs, of use in treatment of neuromuscular disorders, have been hard to identify. However, identification of nAChR allosteric modulator binding sites has been facilitated by using drugs developed as photoreactive GABAR modulators. Recently, 1-methyl-5-allyl-5-(-trifluoromethyl-diazirinylphenyl) barbituric acid (TFD-MPAB), an anesthetic and GABAR potentiator, has been shown to inhibit αβγδ nAChRs, binding in the ion channel and to a γ-α subunit interface site similar to its GABAR intersubunit binding site. In contrast, 1-methyl-5-propyl-5-(-trifluoromethyl-diazirinylphenyl) barbituric acid (TFD-MPPB) acts as a convulsant and GABAR inhibitor. Photolabeling studies established that TFD-MPPB binds to the same GABAR intersubunit binding site as TFD-MPAB, but with negative rather than positive energetic coupling to GABA binding. We now show that TFD-MPPB binds with the same state (agonist) dependence as TFD-MPAB within the nAChR ion channel, but it does not bind to the intersubunit binding site. Rather, TFD-MPPB binds to intrasubunit sites within the α and δ subunits, photolabeling αVal-218 (αM1), δPhe-232 (δM1), δThr-274 (δM2), and δIle-288 (δM3). Propofol, a general anesthetic that binds to GABAR intersubunit sites, inhibited [H]TFD-MPPB photolabeling of these nAChR intrasubunit binding sites. These results demonstrate that in an nAChR, the subtle difference in structure between TFD-MPPB and TFD-MPAB (chirality; 5-propyl 5-allyl) determines selectivity for intra- intersubunit sites, in contrast to GABARs, where this difference affects state dependence of binding to a common site.